The Role of Bone Marrow-Derived Mesenchymal Stromal Cells and Hesperidin in Ameliorating Nephrotoxicity Induced by Cisplatin in Male Wistar Rats

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and antioxidants opened the way for many effective therapeutic experiments against damaged organs like kidneys. Nephrotoxicity is the main complication of chemotherapeutic drugs. Therefore, the present study aimed to investigate the efficacy of BM-MSCs and hesperidin to treat cisplatin-induced nephrotoxicity in rats. Fifty rats were divided into five equal groups of 10 each. Group-I served as a control group, group-II received a single dose of cisplatin (7.5 mg/kg) intraperitoneally to induce nephrotoxicity, group-III received a daily dose of hesperidin (40 mg/kg) orally for four weeks, and on the 5th day cisplatin was administered an hour before hesperidin administration. Group-IV consisted of cisplatin-treated rats that were intravenously injected with 1х106 BM-MSCs cells/rat once per week. Group V contained cisplatin-treated rats that received a combination of hesperidin and BM-MSCs with the same dosage regimes. After four weeks, serum and kidney samples were collected for biochemical, histological, and immunohistochemical examinations were performed. Cisplatin administered rats showed deteriorated biochemical parameters and severe degenerative changes in renal tissue. Both single and combined hesperidin and BM-MSCs treatments restored the renal biochemical parameters. Histologically, the renal tissues significantly improved in the BM-MSCs treated group in comparison with the hesperidin treated group. Moreover, combined treatment (i.e., group V) showed complete restoration of the normal architecture in the renal tissue. Our data suggest that the combined treatment of BM-MSCs and hesperidin has a potent renoprotective efficacy against cisplatin-induced nephrotoxicity rather than the single treatment.

They can also regulate the immune response and secrete potent paracrine factors that promote structural and functional renal recovery. BM-MSCs are considered as free-radical scavenging cells that can reduce oxidative stress by releasing exosomes to prevent ROS accumulation (9).
Hesperidin, a natural flavonoid antioxidant, is found in citrus and has antibacterial, anticancer, anti-inflammatory, and anti-apoptotic properties (10). Due to its antioxidant activity it can attenuate oxidative stress by inhibiting free radical formation (11). The hesperidin antioxidant activity was attributed to its hydrogen-donating properties, which neutralize hydroxyl and superoxide free radicals. As a result, much interest has been drawn to hesperidin as a renoprotective compound against cisplatin-induced renal damage (12).
The current study aimed to assess the effects of BM-MSCs and hesperidin treatment alone or as a combination treatment to ameliorate cisplatininduced nephrotoxicity and to restore renal structure and function in Wistar rats.

BM-MSCs isolation, preparation, and culturing
The BM-MSCs were isolated as previously described (13,14). Briefly, male albino Wistar rats (4 weeks old, 60-80 g body weight) were used to isolate BM-MSCs. Rats were euthanized by an overdose of inhaled isoflurane anesthetic (15) pellet was re-suspended in 1 ml DMEM, and the cell viability was tested again.

Morphological examination of BM-MSCs
BM-MSCs were examined under the inverted microscope to identify their characteristic fusiform shape and well-developed cytoplasmic processes (18).

Determination of serum sodium and potassium levels
Serum sodium and potassium levels were determined by Spectrum-Diagnostics Sodium reagents (Spectrum Diagnostics, Egypt) using the colorimetric method (22). histological changes in more than 75% studied microscopic domains (24).

Immunohistochemistry
The p53 protein immunohistochemical staining was performed using the avidin-biotinperoxidase method (25). Briefly, the tissue sections were deparaffinized and rehydrated using xylene and alcohol, respectively, and were then boiled in

Statistical analysis
The obtained data were expressed as mean were considered as significant.

Results
RT-qPCR was performed to characterize the isolated BM-MSCs. BM-MSCs showed a high

Immunohistochemistry of p53
The p53 protein immunohistochemistry of renal tissues showed that most renal corpuscular and tubular epithelial cells of the control group (group I) exhibited a weak reaction for the p53 ( Figure 5A). However, in response to nephrotoxicity induced by cisplatin treatment (group II), a progressive and robust reaction to the p53 was noticed, with a widespread brown color in the affected renal cells ( Figure 5B). The immunostained sections of the hesperidin-treated rats (group III) showed moderate reaction ( Figure   5C), while the BM-MSCs treated rats (group IV) renal tissues reacted weakly in comparison with the cisplatin group ( Figure 5D). The combined weakly to the p53 antibody ( Figure 5E). Figure 5F shows

Discussion
The current study revealed that hesperidin,  New strategies are directed to enhance the treatment efficacy of BM-MSCs (38), and to overcome the low survival ratio of the transplanted stromal cells induced by their insufficient resistance against the oxidative and inflammatory stresses at the injured sites (31). Due to their insufficient resistance to oxidative and inflammatory stresses at the damaged sites, the transplanted stromal cells have low survival ratios, which is the crucial problem affecting stromal cell therapy (31). Hence, our results further confirm that the antioxidant pretreatments is able to significantly increase the stromal cell longevity, viability, and repair efficacy (6). Studies showed that the treatment of diabetic rats with hesperidin and BM-MSCs together was the most potent approach in ameliorating deteriorated lipid profile, heart and kidney functions (13). Also, antioxidant preconditioning could effectively improve the therapeutic effect of adipose-derived mesenchymal stem cell therapy for liver fibrosis (39).
Interestingly, the current study showed that the pre-treatment of cisplatin-treated rats with hesperidin antioxidant followed by BM-MSCs injection remarkably improved the kidney function and renal architecture. All treatments, either separately or in combination, improved the renal function parameters after cisplatin administration with no significant differences. However, according to our histological assessment, we noticed a substantial restoration of the renal architecture compared to the single treatment, which became similar to the control group (40).
Therefore, the current study revealed that combined treatment of hesperidin and BM-MSCs has the most significant restoring effect against the cisplatin nephrotoxicity than using them separately.
In conclusion, the cisplatin-induced nephrotoxicity and biochemical renal parameters